Melanocortin receptor-specific peptides for treatment of female sexual dysfunction

ABSTRACT

Methods for use of a melanocortin receptor agonist cyclic peptide of the formula 
                         
where R, x and y are as defined in the specification, and compositions and formulations including the peptide of the foregoing formula, for preventing, ameliorating or treating female sexual dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.12/952,238, entitled “Melanocortin Receptor-Specific Peptides forTreatment of Sexual Dysfunction,” filed on Nov. 23, 2010, now U.S. Pat.No. 8,487,073, issued on Jul. 16, 2013. U.S. Ser. No. 12/952,238 is acontinuation of International Application No. PCT/US09/46571, publishedas International Publication No. WO 2009/152079, entitled “MelanocortinReceptor-Specific Peptides for Treatment of Sexual Dysfunction”, filedon Jun. 8, 2009, which in turn claims priority to and the benefit of thefiling of U.S. Provisional Patent Application Ser. No. 61/059,910entitled “Melanocortin Receptor-Specific Peptides for Treatment ofSexual Dysfunction”, filed on Jun. 9, 2008. The specification and claimsof each of the foregoing are incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention (Technical Field)

The present invention relates to melanocortin receptor-specific cyclicpeptides which may be used in the treatment of melanocortinreceptor-mediated diseases, indications, conditions and syndromes.

2. Description of Related Art

The following discussion refers to a number of publications by author(s)and year of publication, and that due to recent publication datescertain publications are not to be considered as prior art vis-a-vis thepresent invention. Discussion of such publications herein is given formore complete background and is not to be construed as an admission thatsuch publications are prior art for patentability determinationpurposes.

A family of melanocortin receptor types and subtypes have beenidentified, including melanocortin-1 receptors (MC1-R) expressed onnormal human melanocytes and melanoma cells, melanocortin-2 receptors(MC2-R) for ACTH (adrenocorticotropin) expressed in cells of the adrenalgland, melanocortin-3 and melanocortin-4 receptors (MC3-R and MC4-R)expressed primarily in cells in the hypothalamus, mid-brain andbrainstem, and melanocortin-5 receptors (MC5-R), expressed in a widedistribution of peripheral tissues.

Significant work has been done in determining the structure ofmelanocortin receptors, including both the nucleic acid sequencesencoding for the receptors and the amino acid sequences constituting thereceptors. MC4-R is a G protein-coupled, 7-transmembrane receptor thatis believed to be expressed primarily in the brain.

Peptides specific for MC4-R, and secondarily peptides specific forMC3-R, are believed to be useful in regulation of mammalian energyhomeostasis, including use as agents for attenuating food intake andbody weight gain. MC4-R agonist peptides are believed to be useful fortreating sexual dysfunction, including male erectile dysfunction, andfor decreasing food intake and body weight gain, such as for treatmentof obesity. MC4-R agonist peptides may also be employed for decreasingvoluntary ethanol consumption, treatment of drug addictions, and thelike. Such peptides, as well as MC1-R and MC3-R agonist peptides, mayfurther be employed for treatment of circulatory shock, ischemia,hemorrhagic shock, inflammatory diseases and related diseases,indications, conditions and syndromes. MC4-R antagonist peptides, bycontrast, are believed to be useful for weight gain aid, such as for usein treatment of cachexia, sarcopenia, wasting syndrome or disease, andanorexia. Such peptides may also be employed for treatment of depressionand related disorders.

Melanocortin receptor-specific peptides include cyclica-melanocyte-stimulating hormone (“α-MSH”) analog peptides such asAc-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-NH₂ (SEQ ID NO:1) (See U.S.Pat. Nos. 5,674,839 and 5,576,290) andAc-Nle-cyclo(-Asp-His-D-Phe-Arg-Trp-Lys)-OH (SEQ ID NO:2) (See U.S. Pat.Nos. 6,579,968 and 6,794,489). These and other melanocortinreceptor-specific peptides generally contain the central tetrapeptidesequence of native α-MSH, His⁶-Phe⁷-Arg⁸-Trp⁹ (SEQ ID NO:3), or amimetic or variation thereof, including the substitution of D-Phe forPhe⁷. Other peptides or peptide-like compounds asserted to be specificfor one or more melanocortin receptors are disclosed in U.S. Pat. Nos.5,731,408, 6,054,556, 6,350,430, 6,476,187, 6,600,015, 6,613,874,6,693,165, 6,699,873, 6,887,846, 6,951,916, 7,008,925, and 7,176,279; inU.S. published patent application Publication Nos. 2001/0056179,2002/0143141, 2003/0064921, 2003/0105024, 2003/0212002, 2004/0023859,2005/0130901, 2005/0187164, 2005/0239711, 2006/0105951, 2006/0111281,2006/0293223, 2007/0027091, 2007/0105759, 2007/0123453, 2007/0244054,and 2008/0039387; and in international patent applications nos. WO98/27113, WO 99/21571, WO 00/05263, WO 99/54358, WO 00/35952, WO00/58361, WO 01/30808, WO 01/52880, WO 01/74844, WO 01/85930, WO01/90140, WO 02/18437, WO 02/26774, WO 03/006604, WO 2004/046166, WO2005/000338, WO 2005/000339, WO 2005/000877, WO 2005/030797, WO2005/060985, WO2006/048449, WO 2006/048450, WO 2006/048451, WO2006/048452, WO 2006/097526, WO 2007/008684, WO 2007/008704, and WO2007/009894.

Notwithstanding the intense scientific and pharmaceutical interest inmelanocortin receptor-specific peptides, evidenced by numerous articlesin the scientific literature and numerous patent applications and issuedpatents, no melanocortin receptor-specific peptide has been approved asa drug for any therapeutic indication. Indeed, there are no reports ofany melanocortin receptor-specific peptide for any therapeuticindication having advanced past Phase II clinical trials. There remainsa significant and substantial need for melanocortin receptor-specificpeptides for use in pharmaceutical applications, and in particular fortreatment of sexual dysfunction. It is against this background that thepresent invention was made.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a cyclic peptide offormula (I):

or a pharmaceutically acceptable salt thereof,

wherein:

-   -   R is —C(═O)—OH or —C(═O)—NH₂;    -   x is 1 or 2; and    -   y is 3 or 4.        The cyclic peptide may be one of formula (II):

Thus the cyclic peptide may be

(SEQ ID NO: 4) Ac-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH₂,(SEQ ID NO: 5) Ac-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH,(SEQ ID NO: 6) Ac-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-NH₂ or(SEQ ID NO: 7) Ac-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-OH.

In another aspect, the present invention provides a pharmaceuticalcomposition comprising a cyclic peptide or pharmaceutically acceptablesalt thereof of the invention and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a melanocortinreceptor-specific peptide-based pharmaceutical composition for use intreatment of melanocortin receptor-mediated diseases, indications,conditions and syndromes.

In another aspect, the present invention provides a peptide-basedmelanocortin receptor-specific pharmaceutical, wherein the peptide is aselective MC4-R ligand, for use in treatment of sexual dysfunction andother MC4-R associated disorders.

In another aspect, the present invention provides peptides which arespecific for MC4-R and which are agonists.

In another aspect, the present invention provides a melanocortinreceptor-specific pharmaceutical for use in treatment sexual dysfunctionwithout substantial adverse cardiovascular effects, including without asubstantial increase in blood pressure.

In another aspect, the present invention provides a specific MC4-Rcyclic peptide that is effective over a significant dose range.

Other aspects and novel features, and the further scope of applicabilityof the present invention will be set forth in part in the detaileddescription to follow, taken in conjunction with the accompanyingdrawings, and in part will become apparent to those skilled in the artupon examination of the following, or may be learned by practice of theinvention. The aspects of the invention may be realized and attained bymeans of the instrumentalities and combinations particularly pointed outin the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and forms a partof the specification, illustrate one or more embodiments of the presentinvention and, together with the description, serves to explain theprinciples of the invention. The drawings are only for the purpose ofillustrating one or more preferred embodiments of the invention and arenot to be construed as limiting the invention.

FIG. 1 is a plot of systolic blood pressure determined in a crossoverstudy using surgically implanted pressure transducers monitored bytelemetry, following administration of vehicle (∘), bremelanotide at adose of 1 μmol/kg by IV injection (□) and the cyclic peptide of Example8.1 at doses of 0.84 μmol/kg (∇) and 3.0 μmol/kg (Δ) by subcutaneousinjection.

FIG. 2 is a plot of systolic blood pressure determined in a crossoverstudy using surgically implanted pressure transducers monitored bytelemetry, following administration of two different lots of the cyclicpeptide of Example 8.4 at doses of 1 mg/kg by subcutaneous injection (∇,Δ) and bremelanotide at a dose of 1 mg/kg by IV injection (□).

DETAILED DESCRIPTION OF THE INVENTION

1.0 Definitions

Before proceeding with the description of the invention, certain termsare defined as set forth herein.

In the sequences given for the peptides according to the presentinvention, the amino acid residues have their conventional meaning asgiven in Chapter 2400 of the Manual of Patent Examining Procedure,8^(th) Ed., published by the United States Patent and Trademark Office.Thus, “Nle” is norleucine, “Asp” is aspartic acid, “His” is histidine,“Phe” is phenylalanine, “Arg” is arginine, “Trp” is tryptophan, and“Lys” is lysine, and so on. It is to be understood that “D” isomers aredesignated by a “D-” before the three letter code or amino acid name,such that for example D-Phe is D-phenylalanine. Amino acid residues notencompassed by the foregoing have the following definitions:

Abbreviation Common Name Side Chain Structure Dab 2,4-diaminobutyricacid

Orn ornithine

The term “Ac” means the acetyl group CH₃—C(═O)—.

An “amide” includes compounds that have a trivalent nitrogen attached toa carbonyl group (—C(═O)—NH₂), such as for example methylamide,ethylamide, propylamide, and the like.

An “amine” includes compounds that contain an amino group (—NH₂).

The term “composition”, as in pharmaceutical composition, is intended toencompass a product comprising the active ingredient(s), and the inertingredient(s) that make up the carrier, as well as any product whichresults, directly or indirectly, from combination, complexation oraggregation of any two or more of the ingredients, or from dissociationof one or more of the ingredients, or from other types of reactions orinteractions of one or more of the ingredients. Accordingly, thepharmaceutical compositions utilized in the present invention encompassany composition made by admixing an active ingredient and one or morepharmaceutically acceptable carriers.

By a melanocortin receptor “agonist” is meant an endogenous substance,drug substance or compound, including a compound such as the peptides ofthe present invention, which can interact with a melanocortin receptorand initiate a pharmacological response, including but not limited toadenyl cyclase activation, characteristic of the melanocortin receptor.For the present invention, a melanocortin receptor agonist which is anagonist at melanocortin-4 receptor (MC4-R) is preferred, but for certainapplications, a melanocortin receptor agonist which is an agonist atboth MC4-R and melanocortin-1 receptor (MC1-R) is preferred, and forother applications a melanocortin receptor agonist which is an agonistat one or more of MC1-R, melanocortin-3 receptor (MC3-R), MC4-R andmelanocortin-5 receptor (MC5-R) is preferred.

By “α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ ID NO:8)and analogs and homologs thereof, including without limitationNDP-α-MSH.

By “NDP-α-MSH” is meant the peptideAc-Ser-Tyr-Ser-Nle-Glu-His-D-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂ (SEQ IDNO:9) and analogs and homologs thereof.

By “EC₅₀” is meant the molar concentration of an agonist, including apartial agonist, which produced 50% of the maximum possible response forthat agonist. By way of example, a test compound which, at aconcentration of 72 nM, produces 50% of the maximum possible responsefor that compound as determined in a cAMP assay in an MC4-R cellexpression system has an EC₅₀ of 72 nM. Unless otherwise specified, themolar concentration associated with an EC₅₀ determination is innanomoles per liter (nM).

By “Ki (nM)” is meant the equilibrium inhibitor dissociation constantrepresenting the molar concentration of a competing compound that bindsto half the binding sites of a receptor at equilibrium in the absence ofradioligand or other competitors. In general, the numeric value of theKi is inversely correlated to the affinity of the compound for thereceptor, such that if the Ki is low, the affinity is high. Ki may bedetermined using the equation of Cheng and Prusoff (Cheng Y., Prusoff W.H., Biochem. Pharmacol. 22: 3099-3108, 1973):

${Ki} = \frac{{EC}_{50}}{1 + \frac{\lbrack{ligand}\rbrack}{K_{D}}}$where “ligand” is the concentration of radioligand and K_(D) is aninverse measure of receptor affinity for the radioligand which produces50% receptor occupancy by the radioligand. Unless otherwise specified,the molar concentration associated with a Ki determination is in nM. Kimay be expressed in terms of specific receptors (e.g., MC1-R, MC3-R,MC4-R or MC5-R) and specific ligands (e.g., α-MSH or NDP-α-MSH).

By “inhibition” is meant the percent attenuation, or decrease inreceptor binding, in a competitive inhibition assay compared to a knownstandard. Thus, by “inhibition at 1 μM (NDP-α-MSH)” is meant the percentdecrease in binding of NDP-α-MSH by addition of a determined amount ofthe compound to be tested, such as 1 μM of a test compound, such asunder the assay conditions hereafter described. By way of example, atest compound that does not inhibit binding of NDP-α-MSH has a 0%inhibition, and a test compound that completely inhibits binding ofNDP-α-MSH has a 100% inhibition. Typically, as described hereafter, aradio assay is used for competitive inhibition testing, such as withI¹²⁵-labeled NDP-α-MSH, or a lanthanide chelate fluorescent assay, suchas with Eu-NDP-α-MSH. However, other methods of testing competitiveinhibition are known, including use of label or tag systems other thanradioisotopes, and in general any method known in the art for testingcompetitive inhibition may be employed in this invention. It may thus beseen that “inhibition” is one measure to determine whether a testcompound attenuates binding of α-MSH to melanocortin receptors.

By “binding affinity” is meant the ability of a compound or drug to bindto its biological target, expressed herein as Ki (nM).

By “intrinsic activity” is meant the maximal functional activityachievable by a compound in a specified melanocortin receptor expressingcell system, such as the maximal stimulation of adenylyl cyclase. Themaximal stimulation achieved by α-MSH or NDP-α-MSH is designated as anintrinsic activity of 1.0 (or 100%) and a compound capable ofstimulating half the maximal activity that of α-MSH or NDP-α-MSH isdesignated as having an intrinsic activity of 0.5 (or 50%). A compoundof this invention that under assay conditions described herein has anintrinsic activity of 0.7 (70%) or higher is classified as an agonist, acompound with intrinsic activity between 0.1 (10%) and 0.7 (70%) isclassified as a partial agonist, and a compound with intrinsic activitybelow 0.1 (10%) is classified as inactive or having no intrinsicactivity. In one aspect, the cyclic peptides of the present inventionmay generally be characterized as a partial agonist at MC4-R withrespect to α-MSH or NDP-α-MSH.

In general, “functional activity” is a measure of the signaling of areceptor, or measure of a change in receptor-associated signaling, suchas a melanocortin receptor, and in particular MC4-R or hMC4-R, uponstimulation by a compound. Melanocortin receptors initiate signaltransduction through activation of heterotrimeric G proteins. In oneaspect, melanocortin receptors signal through Gα_(s), which catalyzesproduction of cAMP by adenylyl cyclase. Thus determination ofstimulation of adenylyl cyclase, such as determination of maximalstimulation of adenylyl cyclase, is one measure of functional activity,and is the primary measure exemplified herein. However, it is to beunderstood that alternative measures of functional activity may beemployed in the practice of this invention, and are specificallycontemplated and included within the scope of this invention. Thus, inone example intracellular free calcium may be measured, such as reportedby and using the methods disclosed in Mountjoy K. G. et al.,Melanocortin receptor-medicated mobilization of intracellular freecalcium in HEK293 cells. Physiol Genomics 5:11-19, 2001, or Kassack M.U. et al., Functional screening of G protein-coupled receptors bymeasuring intracellular calcium with a fluorescence microplate reader.Biomol Screening 7:233-246, 2002. It is also possible to measureactivation by measurement of the production of inositol triphosphate ordiacylglycerol from phosphatidylinositol 4,5-biphosphate, such as by useof radioassays. Yet another measure of functional activity is receptorinternalization, resulting from activation of regulatory pathways, suchas using the methods disclosed in Nickolls S. A. et al., Functionalselectivity of melanocortin 4 receptor peptide and nonpeptide agonists:evidence for ligand specific conformational states. J Pharm ExperTherapeutics 313:1281-1288, 2005. Yet another measure of functionalactivity is the exchange, and exchange rate, of nucleotides associatedwith activation of a G protein receptor, such as the exchange of GDP(guanosine diphosphate) for GTP (guanosine triphosphase) on the Gprotein α subunit, which may be measured by any number of means,including a radioassay using guanosine 5′-(γ-[³⁵S]thio)-triphosphate, asdisclosed in Manning D. R., Measures of efficacy using G proteins asendpoints: differential engagement of G proteins through singlereceptors. Mol Pharmacol 62:451-452, 2002. Various gene-based assayshave been developed for measuring activation of G-coupled proteins, suchas those disclosed in Chen W. et al., A colorimetric assay frommeasuring activation of Gs- and Gq-coupled signaling pathways. AnalBiochem 226:349-354, 1995; Kent T. C. et al., Development of a genericdual-reporter gene assay for screening G-protein-coupled receptors.Biomol Screening, 5:437-446, 2005; or Kotarsky K. et al., Improvedreceptor gene assays used to identify ligands acting on orphanseven-transmembrane receptors. Pharmacology & Toxicology 93:249-258,2003. The colorimetric assay of Chen et al. has been adapted for use inmeasuring melanocortin receptor activation, as disclosed in Hruby V. J.et al., Cyclic lactam α-melanocortin analogues ofAc-Nle⁴-cyclo[Asp⁵,D-Phe⁷,Lys¹⁰] α-melanocyte-stimulatinghormone-(4-10)-NH₂ with bulky aromatic amino acids at position 7 showshigh antagonist potency and selectivity at specific melanocortinreceptors. J Med Chem 38:3454-3461, 1995. In general, functionalactivity may be measured by any method, including methods of determiningactivation and/or signaling of a G-coupled receptor, and furtherincluding methods which may be hereafter developed or reported. Each ofthe foregoing articles, and the methods disclosed therein, isincorporated here by reference as if set forth in full.

The terms “treat,” “treating” and “treatment,” as used herein,contemplate an action that occurs while a patient is suffering from thespecified disease or disorder, which reduces the severity of the diseaseor disorder.

As used herein, the term “therapeutically effective amount” means theamount of a compound including a peptide of the invention that willelicit a biological or medical response in the mammal that is beingtreated by a medical doctor or other clinician.

As used herein, the term “prophylactically effective” or “preventive”means the amount of a compound including a peptide of the invention thatwill prevent or inhibit affliction or mitigate affliction of a mammalwith a medical condition that a medical doctor or other clinician istrying to prevent, inhibit, or mitigate before a patient begins tosuffer from the specified disease or disorder.

“Sexual dysfunction” means any condition that inhibits or impairs normalsexual function, including coitus. The term is not limited tophysiological conditions, and includes psychogenic conditions orperceived impairment without a formal diagnosis of pathology ordisorder. Sexual dysfunction includes erectile dysfunction in a malemammal and female sexual dysfunction in a female mammal.

“Erectile dysfunction” is a disorder involving the failure of a malemammal to achieve functional erection, ejaculation, or both. Erectiledysfunction is accordingly synonymous with impotence, and includes theinability to attain or sustain an erection of sufficient rigidity forcoitus. Symptoms of erectile dysfunction include an inability to achieveor maintain an erection, ejaculatory failure, premature ejaculation, orinability to achieve an orgasm. An increase in erectile dysfunction isoften associated with age or may be caused by a physical disease or as aside-effect of drug treatment.

“Female sexual dysfunction” is a disorder including sexual arousaldisorder. The term “sexual arousal disorder” includes a persistent orrecurrent failure to attain or maintain the lubrication-swellingresponse of sexual excitement until completion of sexual activity.Sexual dysfunction in females can also include inhibited orgasm anddyspareunia, which is painful or difficult coitus. Female sexualdysfunction includes, but is not limited to, a number of categories ofdiseases, conditions and disorders including hypoactive sexual desiredisorder, sexual anhedonia, sexual arousal disorder, dyspareunia andvaginismus. Hypoactive sexual desire disorder includes a disorder inwhich sexual fantasies and desire for sexual activity are persistentlyor recurrently diminished or absent, causing marked distress orinterpersonal difficulties. Hypoactive sexual desire disorder can becaused by boredom or unhappiness in a long-standing relationship,depression, dependence on alcohol or psychoactive drugs, side effectsfrom prescription drugs, or hormonal deficiencies. Sexual anhedoniaincludes decreased or absent pleasure in sexual activity. Sexualanhedonia can be caused by depression, drugs, or interpersonal factors.Sexual arousal disorder can be caused by reduced estrogen, illness, ortreatment with diuretics, antihistamines, antidepressants, orantihypertensive agents. Dyspareunia and vaginismus are sexual paindisorders characterized by pain resulting from penetration and may becaused, for example, by medications which reduce lubrication,endometriosis, pelvic inflammatory disease, inflammatory bowel diseaseor urinary tract problems.

2.0 Clinical Indications and Utility

The compositions and methods disclosed herein can be used for bothmedical applications and animal husbandry or veterinary applications.Typically, the methods are used in humans, but may also be used in othermammals. The term “patient” is intended to denote a mammalianindividual, and is so used throughout the specification and in theclaims. The primary applications of the present invention involve humanpatients, but the present invention may be applied to laboratory, farm,zoo, wildlife, pet, sport or other animals.

Peptides, compositions and methods of the present invention may beemployed for the treatment of sexual dysfunction, including both maleerectile dysfunction and female sexual dysfunction. In one particularembodiment, the peptides, compositions and methods of the presentinvention are used in male patients to increase erectile function,including but not limiting to increasing erectile function so as topermit vaginal intercourse. In another particular embodiment, thepeptides, compositions and methods of the present invention are used totreat female sexual dysfunction, including but not limited to anincrease in arousal success rate, desire success rate, levels of arousaland desire. For female sexual dysfunction, endpoints may, but need not,be determined by any of a number of validated instruments, including butnot limited to the Female Sexual Distress Scale, Female Sexual EncounterProfile, Female Sexual Function Index, and Global AssessmentQuestionnaire. Patients treated for female sexual dysfunction may bepremenopausal women or postmenopausal women.

3.0 Combination Therapy for Certain Indications

The peptides, compositions and methods of the present invention may beused for treatment of any of the foregoing diseases, indications,conditions or syndromes, or any disease, indication, condition orsyndrome which is melanocortin receptor mediated, by administration incombination with one or more other pharmaceutically active compounds.Such combination administration may be by means of a single dosage formwhich includes both a peptide of the present invention and one moreother pharmaceutically active compounds, such single dosage formincluding a tablet, capsule, spray, inhalation powder, injectable liquidor the like. Alternatively, combination administration may be by meansof administration of two different dosage forms, with one dosage formcontaining a peptide of the present invention, and the other dosage formincluding another pharmaceutically active compound. In this instance,the dosage forms may be the same or different. Without meaning to limitcombination therapies, the following exemplifies certain combinationtherapies which may be employed.

3.1 Combination Therapy for Sexual Dysfunction.

It is possible and contemplated to use cyclic peptides of the presentinvention in combination with other drugs or agents for treatment ofsexual dysfunction. These other drugs and agents may include agents thatinduce erectile activity, including phosphodiesterase-5 (PDE-5)inhibitors, testosterone, prostaglandin and the like. In a preferredembodiment of the invention, cyclic peptides of the invention are usedin combination with a therapeutically effective amount of acyclic-GMP-specific phosphodiesterase inhibitor or an alpha-adrenergicreceptor antagonist. The teachings and disclosure of U.S. Pat. No.7,235,625 entitled “Multiple Agent Therapy for Sexual Dysfunction” areincorporated here by reference as if set forth in full.

The present invention thus provides methods of treating sexualdysfunction, the methods comprising the step of administering to thepatient having or at risk of having sexual dysfunction a therapeuticallyeffective amount of a cyclic peptide of the present invention incombination with a therapeutically effective amount of a second sexualdysfunction pharmaceutical agent. The cyclic peptide of the presentinvention may be administered simultaneously with, prior to orsubsequent to administration with a therapeutically effective amount ofa second sexual dysfunction pharmaceutical agent. Preferably the peptideof the present invention is administered within one hour, preferablywithin less than one-half hour, of administration of a therapeuticallyeffective amount of a second sexual dysfunction pharmaceutical agent.However, for certain forms of combination therapy, such as for examplein combination with a therapeutically effective amount of a hormone orhormone-related sexual dysfunction pharmaceutical agent, the hormone orhormone-related sexual dysfunction pharmaceutical agent may beadministered on an independent schedule, such that there is no set orspecific temporal relationship between administration of the peptide ofthe present invention and the hormone or hormone-related sexualdysfunction pharmaceutical agent. Thus, for example, the hormone orhormone-related sexual dysfunction pharmaceutical agent may beadministered on a daily or other dose, or by means of patches or othercontinuous administration schedules, with administration of the peptideof the present invention when desired or needed by the patient.

The present invention thus provides methods of treating sexualdysfunction, the methods comprising the step of administering to apatient having or at risk of having sexual dysfunction a therapeuticallyeffective amount of a cyclic peptide of the present invention incombination with another compound that is useful in the treatment ofsexual dysfunction. In a preferred embodiment of combination therapy thesexual dysfunction is female sexual dysfunction. In an especiallypreferred embodiment of combination therapy the sexual dysfunction iserectile dysfunction.

The present invention also provides pharmaceutical compositions thatcomprise a cyclic peptide of the present invention and a second compounduseful for the treatment of sexual dysfunction. In an embodiment of thecomposition, the additional compounds useful for the treatment of sexualdysfunction are preferably selected from but not limited to the groupconsisting of a phosphodiesterase inhibitor; a cyclic-GMP-specificphosphodiesterase inhibitor; prostaglandins; apomorphine; oxytocinmodulators; a-adrenergic antagonists; androgens; selective androgenreceptor modulators (SARMs); buproprion; vasoactive intestinal peptide(VIP); neutral endopeptidase inhibitors (NEP); and neuropeptide Yreceptor antagonists (NPY).

In an embodiment of the method and composition, the second sexualdysfunction pharmaceutical agent is testosterone.

In another embodiment of combination therapy, the second sexualdysfunction pharmaceutical agent is a type V phosphodiesterase (PDE-5)inhibitor. For example, the PDE-5 inhibitor may be Viagra®, a brand ofsildenafil, Levitra®, a brand of monohydrochloride salt of vardenafil,or Clalis®, a brand of tadalafil. Other PDE-5 inhibitors are disclosedin U.S. Pat. No. 7,235,625, issued Jun. 22, 2007, and entitled “MultipleAgent Therapy for Sexual Dysfunction”, incorporated here by reference.

In another embodiment of the composition above, the compound useful forthe treatment of sexual dysfunction is an estrogen agonist/antagonist.In one embodiment, the estrogen agonist/antagonist is(−)-cis-6-phenyl-5-[-4-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-5,6,7,8-tetrahydro-napththalene-2-ol(also known as lasofoxifene) or an optical or geometric isomer thereof;a pharmaceutically acceptable salt, N-oxide, ester, quaternary ammoniumsalt; or a prodrug thereof. More preferably, the estrogenagonist/antagonist is in the form of a D-tartrate salt.

In yet another embodiment of the composition above, the estrogenagonist/antagonist is selected from the group consisting of tamoxifen,4-hydroxy tamoxifen, raloxifene, droloxifene, toremifene, centchroman,idoxifene,6-(4-hydroxy-phenyl)-5-[-4-(2-piperidine-1-yl-ethoxy)-benzyl]-napthalen-2-ol,{4-[2-(2-aza-bicyclo[2.2.1]hept-2-yl)-ethoxy]-phenyl}-[6-hydroxy-2-(4-hydroxy-phenyl)-benzo[b]thiopehn-3-yl]-methanone,EM-652, EM-800, GW 5368, GW 7604, TSE-424 and optical or geometricisomers thereof; and pharmaceutically acceptable salts, N-oxides,esters, quaternary ammonium salts, and prodrugs thereof.

In yet another embodiment, a cyclic peptide of the present invention maybe used in combination with any known mechanical aids or devices.

The present invention also provides kits for the treatment of sexualdysfunction (including erectile dysfunction), the kits comprising: afirst pharmaceutical composition including a cyclic peptide of thepresent invention; a second pharmaceutical composition comprising asecond compound useful for the treatment of sexual dysfunction; and, acontainer for the first and second compositions.

4.0 Methods of Administration and Use

The method of administration and use varies depending upon thecharacteristic of specific peptides of the present invention, thedisease, indication, condition or syndrome to be treated, and otherfactors known to those in the art. In general, any method ofadministration and use known in the art or hereafter developed may beemployed with the peptides of the present invention. Without limitingthe foregoing, the following methods of administration and use havespecific application for the indicated indications.

Compositions including one or more peptides of the present invention mayadministered by subcutaneous injection. In one aspect, a cyclic peptideof the present invention is formulated for a deep intramuscularinjection, such as in the gluteal or deltoid muscle, of a formulationwith a polyethylene glycol, such as polyethylene glycol 3350, andoptionally one or more additional excipients and preservatives,including but not limited to excipients such as salts, polysorbate 80,sodium hydroxide or hydrochloric acid to adjust pH, and the like. Inanother aspect a cyclic peptide of the present invention is formulatedwith a poly(ortho ester), which may be an auto-catalyzed poly(orthoester) with any of a variable percentage of lactic acid in the polymericbackbone, and optionally one or more additional excipients. In oneaspect poly (D,L-lactide-co-glycolide) polymer is employed. In general,any of a number of injectable and bioerodible polymers, which arepreferably also adhesive polymers, may be employed in a sustainedrelease injectable formulation. Alternatively other sustained releaseformulations may be employed, including formulations permittingsubcutaneous injection, which other formulations may include one or moreof nano/microspheres (such as compositions including PLGA polymers),liposomes, emulsions (such as water-in-oil emulsions), gels, insolublesalts or suspensions in oil The formulation may be such that aninjection is required on a daily, weekly, monthly or other periodicbasis, depending on the concentration and amount of cyclic peptide, thesustained release rate of the materials employed, and other factorsknown to those of skill in the art.

Compositions including one or more peptides of the present invention maybe administered orally in an individual dosage form such as a tablet orcapsule. In one aspect, the individual dosage form includes an entericcoating, and optionally one or more agents to increase uptake, decreaseprotease degradation, increase cellular permeability, and the like.

For sexual dysfunction, in a preferred aspect one or more peptides ofthe present invention are formulated such that it may be administered ondemand, such as about less than one hour, less than two hours or lessthan about four hours prior to anticipated sexual activity. In oneaspect the composition is formulated for subcutaneous injection. Inanother aspect, the composition is formulated for any of a variety oftransdermal routes of administration, including buccal administration,nasal administration, inhalation administration and the like. In anotheraspect the composition is formulated for nasal administration, such as aby means of a metered spray device delivering a volume of from about 20to about 200 μL of an aqueous composition including any of a variety ofother agents, including permeability enhancing agents.

5.0 Methods of Making

In general, the peptides of the present invention may be synthesized bysolid-phase synthesis and purified according to methods known in theart. Any of a number of well-known procedures utilizing a variety ofresins and reagents may be used to prepare the peptides of the presentinvention.

The cyclic peptides of the present invention may be readily synthesizedby known conventional procedures for the formation of a peptide linkagebetween amino acids. Such conventional procedures include, for example,any solution phase procedure permitting a condensation between the freealpha amino group of an amino acid or residue thereof having itscarboxyl group and other reactive groups protected and the free primarycarboxyl group of another amino acid or residue thereof having its aminogroup or other reactive groups protected. In a preferred conventionalprocedure, the cyclic peptides of the present invention may besynthesized by solid-phase synthesis and purified according to methodsknown in the art. Any of a number of well-known procedures utilizing avariety of resins and reagents may be used to prepare the peptides ofthe present invention.

The process for synthesizing the cyclic peptides may be carried out by aprocedure whereby each amino acid in the desired sequence is added oneat a time in succession to another amino acid or residue thereof or by aprocedure whereby peptide fragments with the desired amino acid sequenceare first synthesized conventionally and then condensed to provide thedesired peptide. The resulting peptide is then cyclized to yield acyclic peptide of the invention.

Solid phase peptide synthesis methods are well known and practiced inthe art. In such methods the synthesis of peptides of the invention canbe carried out by sequentially incorporating the desired amino acidresidues one at a time into the growing peptide chain according to thegeneral principles of solid phase methods. These methods are disclosedin numerous references, including Merrifield, R. B., Solid phasesynthesis (Nobel lecture). Angew Chem 24:799-810 (1985) and Barany etal., The Peptides, Analysis, Synthesis and Biology Vol. 2, Gross, E. andMeienhofer, J., Eds. Academic Press 1-284 (1980).

In chemical syntheses of peptides, reactive side chain groups of thevarious amino acid residues are protected with suitable protectinggroups, which prevent a chemical reaction from occurring at that siteuntil the protecting group is removed. Also common is the protection ofthe alpha amino group of an amino acid residue or fragment while thatentity reacts at the carboxyl group, followed by the selective removalof the alpha amino protecting group to allow a subsequent reaction totake place at that site. Specific protecting groups have been disclosedand are known in solid phase synthesis methods and solution phasesynthesis methods.

Alpha amino groups may be protected by a suitable protecting group,including a urethane-type protecting group, such as benzyloxycarbonyl(Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl(Z—Cl), p-nitrobenzyloxycarbonyl (X—NO₂), p-bromobenzyloxycarbonyl(Z—Br), p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc)and p-methoxybenzyloxycarbonyl (Moz) and aliphatic urethane-typeprotecting groups, such as t-butyloxycarbonyl (Boc),diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl(Alloc). Fmoc are preferred for alpha amino protection.

Guanidino groups may be protected by a suitable protecting group, suchas nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc),adamantyloxycarbonyl, pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) andBoc. Pbf and Pmc are preferred protecting groups for Arg.

The peptides of the invention described herein were prepared using solidphase synthesis, such as by means of a Symphony Multiplex PeptideSynthesizer (Rainin Instrument Company) automated peptide synthesizer,using programming modules as provided by the manufacturer and followingthe protocols set forth in the manufacturer's manual.

Solid phase synthesis is commenced from the C-terminal end of thepeptide by coupling a protected alpha amino acid to a suitable resin.Such starting material is prepared by attaching an alpha amino-protectedamino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang)resin, a 2-chlorotrityl chloride resin or an oxime resin, by an amidebond between an Fmoc-Linker, such asp-[(R,S)-α-[1-(9H-fluor-en-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyaceticacid (Rink linker) to a benzhydrylamine (BHA) resin, or by other meanswell known in the art. Fmoc-Linker-BHA resin supports are commerciallyavailable and generally used when feasible. The resins are carriedthrough repetitive cycles as necessary to add amino acids sequentially.The alpha amino Fmoc protecting groups are removed under basicconditions. Piperidine, piperazine, diethylamine, or morpholine (20-40%v/v) in N,N-dimethylformamide (DMF) may be used for this purpose.

Following removal of the alpha amino protecting group, the subsequentprotected amino acids are coupled stepwise in the desired order toobtain an intermediate, protected peptide-resin. The activating reagentsused for coupling of the amino acids in the solid phase synthesis of thepeptides are well known in the art. After the peptide is synthesized, ifdesired, the orthogonally protected side chain protecting groups may beremoved using methods well known in the art for further derivatizationof the peptide.

Typically, orthogonal protecting groups are used as appropriate. Forexample, the peptides of the invention contain multiple amino acids withan amino group-containing side chain. In one aspect, an Allyl-Allocprotection scheme is employed with the amino acids forming a lactambridge through their side chains, and orthogonal protecting groups,cleavable under different reactive conditions, use for other amino acidswith amino group-containing side chains. Thus, for example,Fmoc-Lys(Alloc)-OH, Fmoc-Orn(Alloc)-OH, Fmoc-Dap(Alloc)-OH,Fmoc-Dab(Alloc)-OH, Fmoc-Asp(OAII)-OH or Fmoc-Glu(OAII)-OH amino acidscan be employed for the positions forming a lactam bridge uponcyclization, while other amino acids with amino group-containing sidechains have a different and orthogonal protecting group, such as withFmoc-Arg(Pbf)-OH, Fmoc-Lys(Pbf)-OH, Fmoc-Dab(Pbf)-OH or the like. Otherprotecting groups may be similarly employed; by way of example and notlimitation, Mtt/OPp (4-methyltrityl/2-phenylisopropyl) can be employedwith the side chains forming a lactam bridge upon cyclization, withorthogonal protecting groups being utilized for other positions that arenot cleavable using conditions suitable for cleavage of Mtt/OPp.

Reactive groups in a peptide can be selectively modified, either duringsolid phase synthesis or after removal from the resin. For example,peptides can be modified to obtain N-terminus modifications, such asacetylation, while on resin, or may be removed from the resin by use ofa cleaving reagent and then modified. Similarly, methods for modifyingside chains of amino acids are well known to those skilled in the art ofpeptide synthesis. The choice of modifications made to reactive groupspresent on the peptide will be determined, in part, by thecharacteristics that are desired in the peptide.

In the peptides of the present invention, in one preferred embodimentthe N-terminus group is modified by introduction of an N-acetyl group.In one aspect, a method is employed wherein after removal of theprotecting group at the N-terminal, the resin-bound peptide is reactedwith acetic anhydride in dichloromethane in the presence of an organicbase, such as diisopropylethylamine. Other methods of N-terminusacetylation are known in the art, including solution phase acetylation,and may be employed.

The peptide can, in one embodiment, be cyclized prior to cleavage fromthe peptide resin. For cyclization through reactive side chain moieties,the desired side chains are deprotected, and the peptide suspended in asuitable solvent and a cyclic coupling agent added. Suitable solventsinclude, for example DMF, dichloromethane (DCM) or1-methyl-2-pyrrolidone (NMP). Suitable cyclic coupling reagents include,for example, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TBTU),2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU),benzotriazole-1-yl-oxy-tris(dimethylamino)phosphoniumhexafluorophosphate(BOP),benzotriazole-1-yl-oxy-tris(pyrrolidino)phosphoniumhexafluorophosphate(PyBOP), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate (TATU),2-(2-oxo-1(2H)-pyridyl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TPTU) or N,N′-dicyclohexylcarbodiimide/1-hydroxybenzotriazole(DCCl/HOBt). Coupling is conventionally initiated by use of a suitablebase, such as N,N-diisopropylethylamine (DIPEA), sym-collidine orN-methylmorpholine (NMM).

The cyclized peptides can then be cleaved from solid phase, using anysuitable reagent, such as ethylamine in DCM or various combinations ofagents, such as trifluoroacetic acid (TFA), tri-isopropylsilane (TIS),dimethoxybenezene (DMB), water and the like. The resulting crude peptideis dried and remaining amino acid side chain protecting groups, if any,are cleaved using any suitable reagent, such as (TFA in the presence ofwater, TIS, 2-mercaptopethane (ME) and/or 1,2-ethanedithiol (EDT). Thefinal product is precipitated by adding cold ether and collected byfiltration. Final purification is by reverse phase high performanceliquid chromatography (RP-HPLC), using a suitable column, such as a C₁₈column, or other methods of separation or purification, such as methodsbased on the size or charge of the peptide, can also be employed. Oncepurified, the peptide can be characterized by any number of methods,such as high performance liquid chromatograph (HPLC), amino acidanalysis, mass spectrometry, and the like.

While synthesis has been described primarily with reference to solidphase Fmoc chemistry, it is to be understood that other chemistries andsynthetic methods may be employed to make the cyclic peptides of theinvention, such as by way of example and not limitation, methodsemploying Boc chemistry, solution chemistry, and other chemistries andsynthetic methods.

6.0 Formulations

Depending on the desired route of administration, the formulation of acomposition including one or more cyclic peptides of the presentinvention may be varied. Thus the formulation may be suitable forsubcutaneous injection, or intravenous injection, for topicalapplications, for ocular applications, for nasal spray applications, forinhalation applications, for other transdermal applications and thelike.

6.1 Salt Form of Cyclic Peptides of the Present Invention.

The cyclic peptides of the present invention may be in the form of anypharmaceutically acceptable salt. The term “pharmaceutically acceptablesalts” refers to salts prepared from pharmaceutically acceptablenon-toxic bases or acids including inorganic or organic bases andinorganic or organic acids. Salts derived from inorganic bases includealuminum, ammonium, calcium, copper, ferric, ferrous, lithium,magnesium, manganic salts, manganous, potassium, sodium, zinc, and thelike. Particularly preferred are the ammonium, calcium, lithium,magnesium, potassium, and sodium salts. Salts derived frompharmaceutically acceptable organic non-toxic bases include salts ofprimary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines, and basic ionexchange resins, such as arginine, betaine, caffeine, choline,N,N′-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol,2-dimethylaminoethanol, ethanolamine, ethylenediamine,N-ethyl-morpholine, N-ethylpiperidine, glucamine, glucosamine,histidine, hydrabamine, isopropylamine, lysine, methylglucamine,morpholine, piperazine, piperidine, polyamine resins, procaine, purines,theobromine, triethylamine, trimethylamine, tripropylamine,tromethamine, and the like.

When the cyclic peptide of the present invention is basic, acid additionsalts may be prepared from pharmaceutically acceptable non-toxic acids,including inorganic and organic acids. Such acids include acetic,benzenesulfonic, benzoic, camphorsulfonic, carboxylic, citric,ethanesulfonic, formic, fumaric, gluconic, glutamic, hydrobromic,hydrochloric, isethionic, lactic, maleic, malic, mandelic,methanesulfonic, malonic, mucic, nitric, pamoic, pantothenic,phosphoric, propionic, succinic, sulfuric, tartaric, p-toluenesulfonicacid, trifluoroacetic acid, and the like. Acid addition salts of thepeptides of the present invention are prepared in a suitable solventfrom the peptide and an excess of an acid, such as hydrochloric,hydrobromic, sulfuric, phosphoric, acetic, trifluoroacetic, citric,tartaric, maleic, succinic or methanesulfonic acid. The acetate,ammonium acetate and trifluoracetic acid salt forms are especiallyuseful. Where the peptides of the present invention include an acidicmoiety, suitable pharmaceutically acceptable salts may include alkalimetal salts, such as sodium or potassium salts, or alkaline earth metalsalts, such as calcium or magnesium salts.

6.2 Pharmaceutical Compositions.

The invention provides a pharmaceutical composition that includes acyclic peptide of the present invention and a pharmaceuticallyacceptable carrier. The carrier may be a liquid formulation, and ispreferably a buffered, isotonic, aqueous solution. Pharmaceuticallyacceptable carriers also include excipients, such as diluents, carriersand the like, and additives, such as stabilizing agents, preservatives,solubilizing agents, buffers and the like, as hereafter described.

The cyclic peptide compositions of the present invention may beformulated or compounded into pharmaceutical compositions that includeat least one cyclic peptide of the present invention together with oneor more pharmaceutically acceptable carriers, including excipients, suchas diluents, carriers and the like, and additives, such as stabilizingagents, preservatives, solubilizing agents, buffers and the like, as maybe desired. Formulation excipients may include polyvinylpyrrolidone,gelatin, hydroxy cellulose, acacia, polyethylene glycol, manniton,sodium chloride and sodium citrate. For injection or other liquidadministration formulations, water containing at least one or morebuffering constituents is preferred, and stabilizing agents,preservatives and solubilizing agents may also be employed. For solidadministration formulations, any of a variety of thickening, filler,bulking and carrier additives may be employed, such as starches, sugars,fatty acids and the like. For topical administration formulations, anyof a variety of creams, ointments, gels, lotions and the like may beemployed. For most pharmaceutical formulations, non-active ingredientswill constitute the greater part, by weight or volume, of thepreparation. For pharmaceutical formulations, it is also contemplatedthat any of a variety of measured-release, slow-release or time-releaseformulations and additives may be employed, so that the dosage may beformulated so as to effect delivery of a peptide of the presentinvention over a period of time.

In general, the actual quantity of cyclic peptides of the presentinvention administered to a patient will vary between fairly wide rangesdepending on the mode of administration, the formulation used, and theresponse desired.

In practical use, the cyclic peptides of the invention can be combinedas the active ingredient in an admixture with a pharmaceutical carrieraccording to conventional pharmaceutical compounding techniques. Thecarrier may take a wide variety of forms depending on the form ofpreparation desired for administration, for example, oral, parenteral(including intravenous), urethral, vaginal, rectal, nasal, buccal,sublingual, or the like. In preparing the compositions for oral dosageform, any of the usual pharmaceutical media may be employed, such as,for example, water, glycols, oils, alcohols, flavoring agents,preservatives, coloring agents and the like in the case of oral liquidpreparations such as, for example, suspensions, elixirs and solutions;or carriers such as starches, sugars, microcrystalline cellulose,diluents, granulating agents, lubricants, binders, disintegrating agentsand the like in the case of oral solid preparations such as, forexample, powders, hard and soft capsules and tablets.

Because of their ease of administration, tablets and capsules representan advantageous oral dosage unit form. If desired, tablets may be coatedby standard aqueous or nonaqueous techniques. The amount of activepeptide in such therapeutically useful compositions is such that aneffective dosage will be obtained. In another advantageous dosage unitform, sublingual constructs may be employed, such as sheets, wafers,tablets or the like.

The tablets, pills, capsules, and the like may also contain a bindersuch as gum tragacanth, acacia, corn starch or gelatin; excipients suchas dicalcium phosphate; a disintegrating agent such as corn starch,potato starch or alginic acid; a lubricant such as magnesium stearate;and a sweetening agent such as sucrose, lactose or saccharin. When adosage unit form is a capsule, it may contain, in addition to materialsof the above type, a liquid carrier such as fatty oil.

Various other materials may be utilized as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar or both. A syrup or elixir may contain, in additionto the active ingredient, sucrose as a sweetening agent, methyl andpropylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor.

Cyclic peptides may also be administered parenterally. Solutions orsuspensions of these active peptides can be prepared in water suitablymixed with a surfactant such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof in oils. These preparations may optionally contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that it may be administered by syringe. The form must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, a polyol, for example glycerol,propylene glycol or liquid polyethylene glycol, suitable mixturesthereof, and vegetable oils.

The cyclic peptides of the present invention may be therapeuticallyapplied by means of nasal administration. By “nasal administration” ismeant any form of intranasal administration of any of the cyclicpeptides of the present invention. The peptides may be in an aqueoussolution, such as a solution including saline, citrate or other commonexcipients or preservatives. The peptides may also be in a dry or powderformulation.

The cyclic peptides of the present invention may be formulated with anyof a variety of agents that increase effective nasal absorption ofdrugs, including peptide drugs. These agents should increase nasalabsorption without unacceptable damage to the mucosal membrane. U.S.Pat. Nos. 5,693,608, 5,977,070 and 5,908,825, among others, teach anumber of pharmaceutical compositions that may be employed, includingabsorption enhancers, and the teachings of each of the foregoing, andall references and patents cited therein, are incorporated by reference.

If in an aqueous solution, the cyclic peptides may be appropriatelybuffered by means of saline, acetate, phosphate, citrate, acetate orother buffering agents, which may be at any physiologically acceptablepH, generally from about pH 4 to about pH 7. A combination of bufferingagents may also be employed, such as phosphate buffered saline, a salineand acetate buffer, and the like. In the case of saline, a 0.9% salinesolution may be employed. In the case of acetate, phosphate, citrate,and the like, a 50 mM solution may be employed. In addition to bufferingagents, a suitable preservative may be employed, to prevent or limitbacteria and other microbial growth. One such preservative that may beemployed is 0.05% benzalkonium chloride.

In an alternative embodiment, cyclic peptides of the present inventionmay be administered directly into the lung. Intrapulmonaryadministration may be performed by means of a metered dose inhaler, adevice allowing self-administration of a metered bolus of a peptide ofthe present invention when actuated by a patient during inspiration. Inone aspect of this embodiment, the cyclic peptide may be in a dried andparticulate form, for example particles between about 0.5 and 6.0 μm,such that the particles have sufficient mass to settle on the lungsurface, and not be exhaled, but are small enough that they are notdeposited on surfaces of the air passages prior to reaching the lung.Any of a variety of different techniques may be used to make dry powdermicroparticles, including but not limited to micro-milling, spray dryingand a quick freeze aerosol followed by lyophilization. Withmicroparticles, the peptides may be deposited to the deep lung, therebyproviding quick and efficient absorption into the bloodstream. Further,with such approach penetration enhancers are not required, as issometimes the case in transdermal, nasal or oral mucosal deliveryroutes. Any of a variety of inhalers can be employed, includingpropellant-based aerosols, nebulizers, single dose dry powder inhalersand multidose dry powder inhalers. Common devices in current use includemetered dose inhalers, which are used to deliver medications for thetreatment of asthma, chronic obstructive pulmonary disease and the like.Preferred devices include dry powder inhalers, designed to form a cloudor aerosol of fine powder with a particle size that is always less thanabout 6.0 μm.

Microparticle size, including mean size distribution, may be controlledby means of the method of making. For micro-milling, the size of themilling head, speed of the rotor, time of processing and the likecontrol the microparticle size. For spray drying, the nozzle size, flowrate, dryer heat and the like control the microparticle size. For makingby means of quick freeze aerosol followed by lyophilization, the nozzlesize, flow rate, concentration of aerosoled solution and the likecontrol the microparticle size. These parameters and others may beemployed to control the microparticle size.

The cyclic peptides of the present invention may be therapeuticallyadministered by means of an injection, typically a deep intramuscularinjection, such as in the gluteal or deltoid muscle, of a time releaseinjectable formulation. In one embodiment, a cyclic peptide of thepresent invention is formulated with a polyethylene glycol, such aspolyethylene glycol 3350, and optionally one or more additionalexcipients and preservatives, including but not limited to excipientssuch as salts, polysorbate 80, sodium hydroxide or hydrochloric acid toadjust pH, and the like. In another embodiment a cyclic peptide of thepresent invention is formulated with a poly(ortho ester), which may bean auto-catalyzed poly(ortho ester) with any of a variable percentage oflactic acid in the polymeric backbone, and optionally one or moreadditional excipients. In one embodiment poly (D,L-lactide-co-glycolide)polymer is employed. In general, any of a number of injectable andbioerodible polymers, which are preferably also adhesive polymers, maybe employed in a time release injectable formulation. The formulationmay be such that an injection is required on a weekly, monthly or otherperiodic basis, depending on the concentration and amount of cyclicpeptide, the bioerosion rate of the polymer, and other factors known tothose of skill in the art.

6.3 Oral Formulations of Peptides of the Present Invention.

In one aspect, the cyclic peptides of the present invention areformulated for oral delivery. The peptide is preferably formulated andmade such that it is encased in an enteric protectant, more preferablysuch that it is not released until the tablet or capsule has transitedthe stomach, and optionally has further transited a portion of the smallintestine. In the context of this application it will be understood thatthe term enteric coating or material refers to a coating or materialthat will pass through the stomach essentially intact but will rapidlydisintegrate in the small intestine to release the active drugsubstance. One enteric coating solution that may be used includescellulose acetate phthalate, and optionally other ingredients such asammonium hydroxide, triacetin, ethyl alcohol, methylene blue, andpurified water. Cellulose acetate phthalate is a polymer that has beenused in the pharmaceutical industry for enterically coating individualdosage forms such as tablets and capsules, and is not soluble in waterat a pH of less than about 5.8. Enteric coatings including celluloseacetate phthalate provide protection against the acidic environment ofthe stomach, but begin to dissolve in environment of the duodenum (pH ofabout 6-6.5), and are completely dissolved by the time the dosage formreaches the ileum (pH of about 7-8). In addition to cellulose acetatephthalate, other enteric coating materials are known and may be usedwith peptides of the present invention, including without limitationhydroxypropylmethylethylcellulose succinate,hydroxypropylmethylcellulose phthalate, polyvinyl acetate phthalate, andmethacrylic acid-methyl methacrylate copolymer. The enteric coatingemployed promotes dissolution of the dosage form primarily at a siteoutside the stomach, and may be selected such that the enteric coatingdissolves at a pH of approximately at least 6.0, more preferable at a pHof from about 6.0 to about 8.0. In one preferred aspect, the entericcoating dissolves and breaks down in the proximity of the ileum.

Any of a variety of permeation enhancers may be employed, to increaseuptake in the intestines upon dissolution of the enteric coating. In oneaspect, permeation enhancers increase either paracellular ortranscellular transport systems. An increase in paracellular transportcan be achieved by opening the tight junctions of the cells; an increasein transcellular transport can be achieved by increasing the fluidity ofthe cell membrane. Representative, non-limiting examples of suchpermeation enhancers include calcium chelators, bile salts (such assodium cholate), and fatty acids. The peptides of the present inventionmay be in an enteric-coated individual dosage form that includes a fattyacid, such as for example oleate, palmitate, stearate, sodium caprate,or conjugated linoleic acid, in an enteric-coated capsule, to increaseparacellular transport.

In one aspect, the individual dosage form, such as a tablet or capsule,optionally further includes common pharmaceutical binders such aspovidone, diluents, glidants, fillers such as microcrystallinecellulose, lubricants such as magnesium stearate, disintegrants such ascroscarmellose sodium, preservatives, colorants and the like in theirusual known sizes and amounts. In some embodiments, peptides orpolypeptides that act as substrates for intestinal proteases are furtheradded.

6.4 Routes of Administration.

If a composition including one or more cyclic peptides of the presentinvention is administered by injection, the injection may beintravenous, subcutaneous, intramuscular, intraperitoneal or other meansknown in the art. The peptides of the present invention may beformulated by any means known in the art, including but not limited toformulation as tablets, capsules, caplets, suspensions, powders,lyophilized preparations, suppositories, ocular drops, skin patches,oral soluble formulations, sprays, aerosols and the like, and may bemixed and formulated with buffers, binders, excipients, stabilizers,anti-oxidants and other agents known in the art. In general, any routeof administration by which the peptides of invention are introducedacross an epidermal layer of cells may be employed. Administration meansmay thus include administration through mucous membranes, buccaladministration, oral administration, dermal administration, inhalationadministration, nasal administration, urethral administration, vaginaladministration, rectal administration and the like.

6.5 Therapeutically Effective Amount.

In general, the actual quantity of cyclic peptide of the presentinvention administered to a patient will vary between fairly wide rangesdepending upon the mode of administration, the formulation used, and theresponse desired. The dosage for treatment is administration, by any ofthe foregoing means or any other means known in the art, of an amountsufficient to bring about the desired therapeutic effect. Thus atherapeutically effective amount includes an amount of a peptide orpharmaceutical composition of the present invention that is sufficientto therapeutically alleviate sexual dysfunction in a patient, or toprevent or delay onset or recurrence of the sexual dysfunction.

In general, the cyclic peptides of the present invention are highlyactive. For example, the cyclic peptide can be administered at about0.1, 0.5, 1, 5, 50, 100, 500, 1000 or 5000 μg/kg body weight, dependingon the specific peptide selected, the desired therapeutic response, theroute of administration, the formulation and other factors known tothose of skill in the art.

7.0 Tests and Assays Employed in Evaluation of the Peptides of thePresent Invention

The melanocortin receptor-specific cyclic peptides of the presentinvention of this invention may be tested by a variety of assay systemsand animal models to determine binding, functional status and efficacy.

7.1 Competitive Inhibition Assay Using [I¹²⁵]-NDP-α-MSH.

A competitive inhibition binding assay is performed using membranehomogenates prepared from HEK-293 cells that express recombinant hMC4-R,hMC3-R, or hMC5-R, and from B-16 mouse melanoma cells (containingendogenous MC1-R). In some instances, HEK-293 cells that expressrecombinant hMC1-R were employed. In the examples that follow, allMC3-R, MC4-R and MC5-R values are for human recombinant receptors. MC1-Rvalues are for B-16 mouse melanoma cells, unless the heading is“hMC1-R”, in which case the value is for human recombinant MC1-R. Assayswere performed in 96 well GF/B Millipore multiscreen filtration plates(MAFB NOB10) pre-coated with 0.5% bovine serum albumin (Fraction V).Membrane homogenates were incubated with 0.2 nM (for hMC4-R) 0.4 nM (forMC3-R and MC5-R) or 0.1 nM (for mouse B16 MC1-R or hMC1-R)[I¹²⁵]-NDP-α-MSH (Perkin Elmer) and increasing concentrations of cyclicpeptides of the present invention in buffer containing 25 mM HEPESbuffer (pH 7.5) with 100 mM NaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.3 mM1,10-phenanthroline, and 0.2% bovine serum albumin. After incubation for60 minutes at 37° C., the assay mixture was filtered and the membraneswashed three times with ice-cold buffer. Filters were dried and countedin a gamma counter for bound radioactivity. Non-specific binding wasmeasured by inhibition of binding of [I¹²⁵]-NDP-α-MSH in the presence of1 μM NDP-α-MSH. Maximal specific binding (100%) was defined as thedifference in radioactivity (cpm) bound to cell membranes in the absenceand presence of 1 μM NDP-α-MSH. Radioactivity (cpm) obtained in thepresence of test compounds was normalized with respect to 100% specificbinding to determine the percent inhibition of [I¹²⁵]-NDP-α-MSH binding.Each assay was conducted in triplicate and the actual mean values aredescribed, with results less than 0% reported as 0%. Ki values forcyclic peptides of the present invention were determined using Graph-PadPrism® curve-fitting software. Reported data herein for binding tohMC1-R and hMC4-R utilized [I¹²⁵]-NDP-α-MSH, as did some data withhMC3-R.

7.2 Competitive Binding Assay Using Eu-NDP-α-MSH

Alternatively, a competitive inhibition binding assay was performedemploying Eu-NDP-α-MSH (PerkinElmer Life Sciences catalog No. AD0225)with determination by time-resolved fluorometry (TRF) of the lanthanidechelate. In comparison studies with [I¹²⁵]-NDP-α-MSH, the same values,within experimental error ranges, were obtained for percent inhibitionand Ki. Typically competition experiments to determine Ki values wereconducted by incubating membrane homogenates prepared from HEK-293 cellsthat express recombinant hMC4-R with 9 different concentrations ofcyclic peptides of the present invention and 1 nM of Eu-NDP-α-MSH in asolution containing 25 mM HEPES buffer with 100 mM NaCl, 2 mM CaCl₂, 2mM MgCl₂, 0.1% BSA, and 0.3 mM 1,10-phenanthroline. After incubation for90 minutes at 37° C., the reaction was stopped by filtration overAcroWell 96-well filter plates (Pall Life Sciences). The filter plateswere washed 4 times with 200 μL of ice-cold phosphate-buffered saline.DELFIA Enhancement solution (PerkinElmer Life Sciences) was added toeach well. The plates were incubated on a shaker for 15 minutes and readat 340 nm excitation and 615 nm emission wavelengths. Each assay wasconducted in duplicate and mean values were utilized. Ki values weredetermined by curve-fitting with Graph-Pad Prism® software using aone-site fixed-slope competition binding model. Reported data herein forbinding to hMC5-R and some data with hMC3-R utilized hMC4-R utilizedEu-NDP-α-MSH.

7.3 Competitive Binding Assay using [I¹²⁵]-AgRP (83-132).

Competitive binding studies using [I¹²⁵]-AgRP (83-132) are carried outusing membrane homogenates isolated from cells that express hMC4-R. Theassays were performed in 96-well GF/B Millipore multiscreen filtrationplates (MAFB NOB10) pre-coated with 0.5% bovine serum albumin (FractionV). The assay mixture contained 25 mM HEPES buffer (pH 7.5) with 100 mMNaCl, 2 mM CaCl₂, 2 mM MgCl₂, 0.3 mM 1,10-phenanthroline, 0.5% bovineserum albumin, membrane homogenates, radioligand [I¹²⁵]-AgRP (83-132)(Perkin Elmer) and increasing concentrations of peptides of the presentinvention in a total volume of 200 μL. Binding was measured atradioligand concentrations of 0.2 nM. After incubating for 1 hour at 37°C., the reaction mixture was filtered and washed with assay buffercontaining 500 mM NaCl. The dried discs were punched out from the plateand counted on a gamma counter. The total binding of the radioligand didnot exceed 10% of the counts added to the reaction mixture. Ki valuesfor cyclic peptides of the present invention were determined usingGraph-Pad Prism® curve-fitting software.

7.4 Assay for Agonist Activity.

Accumulation of intracellular cAMP is examined as a measure of theability of the cyclic peptides of the present invention to elicit afunctional response in HEK-293 cells that express MC4-R. ConfluentHEK-293 cells that express recombinant hMC4-R were detached from cultureplates by incubation in enzyme-free cell dissociation buffer. Dispersedcells were suspended in Earle's Balanced Salt Solution containing 10 mMHEPES (pH 7.5), 1 mM MgCl₂, 1 mM glutamine, 0.5% albumin and 0.3 mM3-isobutyl-1-methyl-xanthine (IBMX), a phosphodiesterase inhibitor. Thecells were plated in 96-well plates at a density of 0.5×10⁵ cells perwell and pre-incubated for 30 minutes. Cells were exposed for 1 hour at37° C. to test cyclic peptides of the present invention dissolved inDMSO (final DMSO concentration of 1%) at a concentration range of0.05-5000 nM in a total assay volume of 200 μL. NDP-α-MSH was used asthe reference agonist. At the end of the incubation period, cells weredisrupted by the addition of 50 μL of lysis buffer (cAMP EIA kit,Amersham) followed by vigorous pipetting. Levels of cAMP in the lysateswere determined using a cAMP EIA kit (Amersham). Data analysis wasperformed by nonlinear regression analysis with Graph-Pad Prism®software. The maximum efficacies of the cyclic peptides of the presentinvention were compared to that achieved by the reference melanocortinagonist NDP-αMSH.

7.5 Food Intake and Body Weight Change.

Change in food intake and body weight is evaluated for selected peptidesadministered by intravenous (IV) or subcutaneous injection routes. MaleSprague-Dawley rats are obtained from Hilltop Lab Animals, Inc.(Scottsdale, Pa.) or other vendors. Animals are individually housed inconventional polystyrene hanging cages and maintained on a controlled 12hour on/off light cycle. Water and pelleted food is provided ad libitum.The rats are dosed IV with vehicle or selected peptides (0.3 to 1.0mg/kg), or dosed subcutaneously with vehicle or selected peptides (dosesup to 30 mg/kg). The changes in body weight and food intake for the 24hour period after dosing are determined. The changes in body weight andfood intake for the 48 hour and 72 hour periods after dosing are alsomeasured to determine reversal of changes in body weight and food intakeeffects back to baseline levels.

7.6 Induction of Penile Erection.

The ability of peptides of the present invention to induce penileerection (PE) in male rats are evaluated with selected peptides. MaleSprague-Dawley rats weighing 250-300 g are kept on a 12 hour on/offlight cycle with food and water ad libitum. All behavioral studies areperformed between 9 a.m. and 4 p.m. Groups of 6-8 rats are administeredpeptides at a variety of doses via an IV route. Immediately aftertreatment, rats are placed into individual polystyrene cages (27 cmlong, 16 cm wide, and 25 cm high) for behavioral observation, typicallyby remote video monitoring. Rats are observed for one hour, and thenumber of yawns, grooming bouts and PEs are recorded in 10-minute bins.

8.0 Peptides of the Invention

The cyclic peptides encompassed within formula (I) contain one or moreasymmetric elements such as stereogenic centers, stereogenic axes andthe like, so that the peptides encompassed within formula (I) can existin different stereoisomeric forms. For both specific and genericallydescribed peptides, including the peptides encompassed within formula(I), all forms of isomers at all chiral or other isomeric centers,including enantiomers and diastereomers, are intended to be coveredherein. The peptides of the invention each include multiple chiralcenters, and may be used as a racemic mixture or an enantiomericallyenriched mixture, in addition to use of the peptides of the invention inenantiopure preparations. Typically, the peptides of the invention willbe synthesized with the use of chirally pure reagents, such as specifiedL- or D-amino acids, using reagents, conditions and methods such thatenantiomeric purity is maintained, but it is possible and contemplatedthat racemic mixtures may be made. Such racemic mixtures may optionallybe separated using well-known techniques and an individual enantiomermay be used alone. In cases and under specific conditions oftemperature, solvents and pH wherein peptides may exist in tautomericforms, each tautomeric form is contemplated as being included withinthis invention whether existing in equilibrium or predominantly in oneform. Thus a single enantiomer of a peptide of formula (I), which is anoptically active form, can be obtained by asymmetric synthesis,synthesis from optically pure precursors, or by resolution of theracemates.

The peptides of formula (II) are specific stereoisomeric forms of thepeptides of formula (I), but the invention should not be construed asbeing limited to the stereoisomeric forms encompassed by formula (II).

The invention is further intended to include prodrugs of the presentpeptides, which on administration undergo chemical conversion bymetabolic processes before becoming active pharmacological peptides. Ingeneral, such prodrugs will be functional derivatives of the presentpeptides, which are readily convertible in vivo into a peptide offormula (I). Prodrugs are any covalently bonded compounds, which releasethe active parent peptide drug of formula (I) in vivo. Conventionalprocedures for the selection and preparation of suitable prodrugderivatives are described, for example, in “Design of Prodrugs”, ed. H.Bundgaard, Elsevier, 1985. Typical examples of prodrugs havebiologically labile protecting groups on a functional moiety, such asfor example by esterification of hydroxyl, carboxyl or amino functions.Thus by way of example and not limitation, a prodrug includes peptidesof formula (I) wherein an ester prodrug form is employed, such as, forexample, lower alkyl esters of the R group of formula (I), such as whereR is —OH, which lower alkyl esters may include from 1-8 carbons in analkyl radical or aralkyl esters which have 6-12 carbons in an aralkylradical. Broadly speaking, prodrugs include compounds that can beoxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated,hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated,phosphorylated or dephosphorylated to produce an active parent peptidedrug of formula (I) in vivo.

The subject invention also includes peptides which are identical tothose recited in formula (I), but for the fact that one or more atomsdepicted in formula (I) are replaced by an atom having an atomic mass ormass number different from the atomic mass or mass number usually foundin nature. Examples of isotopes that can be incorporated into compoundsof the invention include isotopes of hydrogen, carbon, nitrogen andoxygen, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O and ¹⁷O, respectively.Peptides of the present invention and pharmaceutically acceptable saltsor solvates of said compounds which contain the aforementioned isotopesand/or other isotopes of other atoms are within the scope of thisinvention. Certain isotopically-labeled compounds of the presentinvention, for example those into which radioactive isotopes such as ³Hand ¹⁴C are incorporated, may have use in a variety of assays, such asdrug and/or substrate tissue distribution assays. Substitution withheavier isotopes, such as substitution of one or more hydrogen atomswith deuterium (²H), can provide pharmacological advantages in someinstances, including increased metabolic stability. Isotopically labeledpeptides of formula (I) can generally be prepared by substituting anisotopically labeled reagent for a non-isotopically labeled reagent.

8.1 The Peptide of the Following Structure was Synthesized:

This peptide has the amino acid sequenceAc-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH₂. The cyclic peptide ofExample 8.1 was prepared as the acetate (AcOH) and trifluoroacetic acid(TFA) salt forms. The cyclic peptide of Example 8.1 has the molecularformula C₄₈H₇₁N₁₇O₉, and has a calculated molecular weight of 1030.19.The molecular weight of the cyclic peptide of Example 8.1 as the acetatesalt form was 1210.34, and as the TFA salt form was 1372.25.

The cyclic peptide of Example 8.1 was evaluated for binding againstMC1-R, MC3-R, MC4-R and MC5-R in competitive studies using NDP-α-MSH,and was found to be selective for MC4-R, with a Ki value of 4.0 nM atMC4-R (average of four studies), a Ki value of 9 nM for MC1-R (averageof five studies), a Ki value of 150 nM for MC3-R (average of twostudies) and a Ki value of 2270 nM for MC5-R (average of two studies).In functional studies, the cyclic peptide of Example 8.1 was determinedto be an agonist at MC4-R, with intrinsic activity of 91% at MC4-R whereNDP-α-MSH is 100%, and with an EC₅₀ of 0.3 nM (average of five studies).

In rat penile erection studies, using bremelanotide (a non-specificMC4-R agonist of the formulaAc-Nle-cyclo(Asp-His-D-Phe-Arg-Trp-Lys)-OH)) as a positive control, thecyclic peptide of Example 8.1 was found to result in a statisticallysignificant increase in observed spontaneous erections in a rat modelcompared to vehicle controls. Vehicle alone administered by asubcutaneous route resulted in an average of 0.429±0.202 spontaneouserections per rat in one hour (n=7), while subcutaneously administeredcyclic peptide of Example 8.1 at a dose of 0.3 mg/kg resulted in anaverage of 2.286±0.286 spontaneous erections per rat in one hour (n=7)and at a dose of 1.0 mg/kg resulted in an average of 4.571±1.088spontaneous erections per rat in one hour (n=7). The positive control,bremelanotide administered IV at a dose of 1 mg/kg, resulted in anaverage of 4±0.535 spontaneous erections per rat in one hour (n=7).

Surprisingly and advantageously, it was found in rat models of systolicblood pressure, using surgically implanted pressure transducersmonitored by telemetry, that the cyclic peptide of Example 8.1 resultedin less increase of systolic blood pressure than did a dose ofbremelanotide producing comparable results in penile erection studies.FIG. 1 shows the results in crossover studies using 8transducer-implanted animals receiving the cyclic peptide of Example 8.1at doses of 0.84 μmol/kg and 3.0 μmol/kg by subcutaneous injection andbremelanotide at a dose of 1 μmol/kg by IV injection, where in companionstudies penile erections per rat per hour of the same doses and routesof administration showed the same or higher penile erection results withthe cyclic peptide of Example 8.1 at 3.0 μmol/kg than with bremelanotideat 1 μmol/kg. In other studies, including comparisons of the samequantities of the cyclic peptide of Example 8.1 and bremelanotide by thesame routes of administration, substantially similar results wereobtained.

8.2 The Peptide of the Following Structure was Synthesized:

This peptide has the amino acid sequenceAc-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH. The cyclic peptide ofExample 8.2 was prepared as the TFA salt form. The cyclic peptide ofExample 8.2 has the molecular formula C₄₈H₇₀N₁₆O₁₀, and has a calculatedmolecular weight of 1031.17. The molecular weight of the cyclic peptideof Example 8.2 as the TFA salt form was 1373.23.

The cyclic peptide of Example 8.2 was evaluated for binding againstMC1-R, MC3-R, MC4-R and MC5-R in competitive studies using NDP-α-MSH,and was found to be selective for MC4-R, with a Ki value of 57 nM atMC4-R (average of two studies), a Ki value of 234 nM for MC1-R (averageof three studies), a Ki value of 5804 nM for MC3-R (average of twostudies) and a Ki value over 10,000 nM for MC5-R (average of twostudies). In functional studies, the cyclic peptide of Example 8.1 wasdetermined to be an agonist at MC4-R, with intrinsic activity of 91% atMC4-R where NDP-α-MSH is 100%, and with an EC₅₀ of 4 nM (average of fourstudies).

8.3 The Peptide of the Following Structure was Synthesized:

This peptide has the amino acid sequenceAc-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-NH₂. The cyclic peptide ofExample 8.3 was prepared as the TFA salt form. The cyclic peptide ofExample 8.3 has the molecular formula C₄₈H₇₁N₁₇O₉, and has a calculatedmolecular weight of 1030.19. The molecular weight of the cyclic peptideof Example 8.3 as the TFA salt form was 1372.25.

The cyclic peptide of Example 8.3 was evaluated for binding againstMC1-R, MC3-R, MC4-R and MC5-R in competitive studies using NDP-α-MSH,and was found to have a Ki value of 0.65 nM at MC4-R (average of twostudies), a Ki value of 1 nM for MC1-R (average of three studies), a Kivalue of 74 nM for MC3-R (average of two studies) and a Ki value of 300nM for MC5-R (average of two studies). In functional studies, the cyclicpeptide of Example 8.3 was determined to be an agonist at MC4-R, withintrinsic activity of 94% at MC4-R where NDP-α-MSH is 100%, and with anEC₅₀ of 0.3 nM (average of five studies).

8.4 The Peptide of the Following Structure was Synthesized:

This peptide has the amino acid sequenceAc-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-OH. The cyclic peptide ofExample 8.4 was prepared as the acetate and TFA salt forms. The cyclicpeptide of Example 8.4 has the molecular formula C₄₈H₇₀N₁₆O₁₀, and has acalculated molecular weight of 1031.17. The molecular weight of thecyclic peptide of Example 8.4 as the acetate salt form was 1211.32, andas the TFA salt form was 1373.23.

The cyclic peptide of Example 8.4 was evaluated for binding againstMC1-R, MC3-R, MC4-R and MC5-R in competitive studies using NDP-α-MSH,and was found to have a Ki value of 8 nM at MC4-R (average of twostudies), a Ki value of 4 nM for MC1-R (one study), a Ki value of 410 nMfor MC3-R (one study) and a Ki value of 2366 nM for MC5-R (average oftwo studies). In functional studies, the cyclic peptide of Example 8.4was determined to be an agonist at MC4-R, with intrinsic activity of 91%at MC4-R where NDP-α-MSH is 100%, and with an EC₅₀ of 3 nM (average ofnine studies).

In rat penile erection studies, using bremelanotide as a positivecontrol, the cyclic peptide of Example 8.4 was found to result in astatistically significant increase in observed spontaneous erections ina rat model compared to vehicle controls. Vehicle alone administered bya subcutaneous route resulted in an average of 0.429±0.202 spontaneouserections per rat in one hour (n=7), while subcutaneously administeredcyclic peptide of Example 8.4 at a dose of 0.3 mg/kg resulted in anaverage of 2.571±0.685 spontaneous erections per rat in one hour (n=7)and at a dose of 1.0 mg/kg resulted in an average of 5.286±0.918spontaneous erections per rat in one hour (n=7). The positive control,bremelanotide administered IV at a dose of 1 mg/kg, resulted in anaverage of 4±0.535 spontaneous erections per rat in one hour (n=7).

Surprisingly and advantageously, it was found in rat models of systolicblood pressure, using surgically implanted pressure transducersmonitored by telemetry, that the cyclic peptide of Example 8.4 resultedin less increase of systolic blood pressure than did a dose ofbremelanotide producing comparable results in penile erection studies.FIG. 2 shows the results in crossover studies using 8transducer-implanted animals receiving the cyclic peptide of Example 8.4at doses of 1 mg/kg by subcutaneous injection and bremelanotide at adose of 1 mg/kg by IV injection, where in companion studies penileerections per rat per hour of the same doses and routes ofadministration showed the same or higher penile erection results withthe cyclic peptide of Example 8.4 at 1 mg/kg than with bremelanotide at1 mg/kg. In other studies, including comparisons of the same quantitiesof the cyclic peptide of Example 8.4 and bremelanotide by the sameroutes of administration, substantially similar results were obtained.

Although the invention has been described in detail with particularreference to these preferred embodiments, other embodiments can achievethe same results. Variations and modifications of the present inventionwill be obvious to those skilled in the art and it is intended to coverall such modifications and equivalents. The entire disclosures of allreferences, applications, patents, and publications cited above arehereby incorporated by reference.

What is claimed is:
 1. A method for treating female sexual dysfunction,comprising the step of administering a pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a cyclic peptide offormula (I):

including all enantiomers, stereoisomers or diastereoisomers thereof, ora pharmaceutically acceptable salt of any of the foregoing, wherein: Ris —C(═O)—OH or —C(═O)—NH₂; x is 1 or 2; and y is 3 or
 4. 2. The methodof claim 1 wherein the cyclic peptide is of formula (II):


3. The method of claim 1 wherein the cyclic peptide isAc-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-NH₂ (SEQ ID NO:4).
 4. The methodof claim 1 wherein the cyclic peptide isAc-Arg-cyclo(Asp-Dab-D-Phe-Arg-Trp-Lys)-OH (SEQ ID NO:5).
 5. The methodof claim 1 wherein the cyclic peptide isAc-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-NH₂ (SEQ ID NO:6).
 6. The methodof claim 1 wherein the cyclic peptide isAc-Arg-cyclo(Glu-Dab-D-Phe-Arg-Trp-Orn)-OH (SEQ ID NO:7)
 7. The methodof claim 1 wherein the step of administering comprises oral, parenteral,urethral, vaginal, rectal, nasal, buccal, or sublingual administration.8. The method of claim 1, wherein the step of administering comprisessubcutaneous administration.
 9. The method of claim 1, furthercomprising the step of administration of a second sexual dysfunctionpharmaceutical agent.